Composition and method for controlling plant diseases using Pseudomonas chlororaphis strain NCIMB 40616

ABSTRACT

A pure culture of Pseudomonas chlororaphis strain NCIMB 40616 is disclosed. The strain is useful for a biocontrol composition for the control of plant fungal diseases. Further, a culture broth of the strain is disclosed to be useful wherein antipathogenically active metabolites are contained in the culture broth. In addition a method of controlling the plant fungal diseases is disclosed which is carried out by the introduction of an effective dose of the strain into a plant environment infected with fungal diseases. Also carriers and additives are admixed with the strain in order to provide for the composition. The types of pathogenic fungi which may be controlled using the method and composition are of the genera Drechslera, Microdochium, Tilletia or Ustilago.

FIELD OF THE INVENTION

The present invention relates to plant protection products. Morespecifically, the invention relates to a novel strain of the bacterialspecies Pseudomonas chlororaphis and the use of compositions containingthis bacterial strain or antibiotic substances produced by this strainin plant production in order to protect plants against attacks byphytopathogenic microbial agents.

BACKGROUND OF THE INVENTION

Several agents of microbial nature with the ability to induce plantdiseases cause considerable damages, and accordingly economic losses, incrop plants. Many of them attack leaves and/or other aerial plant partsand then usually reach new uninfected crops by airborne spores. Othersare transmitted from one crop generation to the next by being seedborneand several economically important disease-inducing agents are soilborneand reside more or less inactive in the soil until a susceptible crop isgrown.

Procedures exerted for controlling microbial disease-inducing agents incrop production are often costly, but in most crop growing systemseconomically necessary. One widely used method is treatment withbiostatic or biocidal chemicals. They are in most cases applied assprays on growing crops, as seed or root treatments or as soildisinfectants. Other standard methods are breeding for resistance andmanagement of the cropping system itself.

These standard control methods all have some drawbacks. Managing of thecropping system is effective or convenient only for certain diseaseproblems. Also the breeding of crop plants for resistance is possible orsuitable only in certain cases, may take long time and the resistanceobtained may be broken after some time by the appearance of new strainsof the pathogen. Chemical compounds often are highly effective, but theymay give unwanted effects in the environment, require careful handlingas most are risky for human health and they also may become ineffectivewhere resistant pathogen strains develop.

The use of biological control agents or biopesticides may be moreeffective or more preferable than the use of other control methods and,thus, such agents have been extensively tried. Several bacterial andfungal strains are known to inhibit growth of various microbialdisease-inducing agents. To be effective and usable they have to bestable, give reproducible results in the field and there must bepossibilities to apply them under field conditions. To date few havefulfilled these requirements and, thus, have been used as commercialproducts.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a biological control agent useful andeffective for controlling plant pathogen attacks in commercial plantgrowings. A novel strain (MA 342) of the bacteria Pseudomonaschlororaphis showing the desired characteristics is provided. Theisolate was deposited at the National Collections of Industrial andMarine Bacteria Limited (NCIMB), Aberdeen, Scotland on Feb. 14, 1994under the terms of the Budapest Treaty and has received NCIMB AccessionNo. 40616.

The invention also provides a plant disease controlling compositioncontaining as active ingredient the novel strain MA 342 or mutantsthereof with essentially the same characteristics or antipathogenicallyactive metabolites or derivates thereof. Further, the invention providesa method of controlling plant diseases using the novel strain MA 342 ormutants thereof with essentially the same characteristics orantipathogenically active metabolites or derivates thereof.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: This figure shows the fatty acid profile obtained with theMicrobial Identification System (MIDI, Newark Ltd., USA) of thebacterial isolate MA 342.

DETAILED DESCRIPTION OF THE INVENTION

Below follows a characterization of the novel bacterial strain and adescription of preferred methods for strain proliferation and forformulations and applications in the field or in greenhouses. Severalexamples are offered to further illustrate, but not to limit, the methodand composition of the invention.

Characterization of the novel bacterial strain MA 342

Morphological characteristics:

Colony morphology on TSA 10 (10 g Tryptic Soy Broth (Difco Ltd.); 12 gTechnical Agar (Oxoid Ltd.) in 1000 ml distilled water) is round, white,moderately convex colonies that form well visible hyaline crystals inthe agar at high cell densities. It is a Gram negative rod that shows abright 1000 ml distilled water).

Fatty acid analysis:

The fatty acid profile of the bacterium is shown in FIG. 1. Thisanalysis was performed using the Microbial Identification System (MIDILtd., Newark, USA), version 3.7. According to this test program, MA 342is most similar to Pseudomonas chlororaphis, with a matching index of0.705.

    ______________________________________    Biochemical characteristics    Characteristics tested    in API 20 NE* rapid test                       Reaction of isolate MA 342    ______________________________________    Nitrate reduction  -    Indole production  -    Acid from glucose  -    Arginine dihydrolase                       +    Urease             -    Esculin hydrolysis -    Gelatin hydrolysis +    B-galactosidase    -    Glucose assimilation                       +    Arabinose assimilation                       -    Mannose assimilation                        -?    Mannitol assimilation                       +    N-acetyl-glucosamine assimilation                       -    Maltose assimilation                       -    Gluconate assimilation                       +    Caprate assimilation                       -    Adipate assimilation                       -    Malate assimilation                       +    Citrate assimilation                       +    Phenyl-acetate assimilation                       -    Cytochrome oxidase +    Characteristics tested    in additional tests    Levan production   -    Xylose assimilation                        -?    Sorbitol assimilation                        +?    ______________________________________     *API System Ltd., France

Preferred methods for strain proliferation and for formulations andapplications in the field or in greenhouses

Quantities of the active strain is best obtained by a fermentationprocess that comprises inoculating a sample of a pure culture of thestrain into a liquid shake culture or in a fermentor containing asuitable fermentation medium. The strain may also be grown on a sterilesurface, e.g. an agar surface, and when grown out, the cells may besuspended in water or other liquid media known in the art. Growing mediamay in principle be any bacterial growth medium known in the art. Thefermentation is carried out until a sufficient concentration of cells,e.g. about 5-10⁹ cfu (colony forming units)/ml for liquid cultures, isobtained. The so obtained fermentation broth or bacterial suspension maybe employed as such for use in plant protection, or they may be treatedor formulated before being used.

In one type of treatment the bacterial cells in the fermentation brothmay be killed, e.g. by heating, or centrifuged down and the resultingbroth or supernatant, containing bacterial metabolites, may be used forplant protection purposes, with or without prior purification and/orconcentration. Bacterial suspensions and fermentation broths may also behomogeneously mixed with one or more compounds or groups of compoundsknown in the art, provided such compounds are compatible with thebacterial strains or its antipathogenically active metabolites orderivates of these. Suitable compounds may be powdery additives or solidcarriers, such as talcum, kaolin, bentonite or montmorillonite, wettablepowders known in the art, carbon source nutrients (such as glucose,sucrose and fructose) or complex bacterial nutrients (such as yeastextract, bacteriological peptone and tryptone), metal salts, salts fromfatty acids, fatty acid esters, ionic or non-ionic surfactants, plantnutrients, plant growth regulators, fungicides, insecticides,bactericides and the like. Bacterial suspensions and fermentation brothsmay also be dried or freeze-dried prior to or after being mixed withsuitable compounds and the resulting product used for plant protection.A suitable way of drying is for example air drying of vermiculitesupplied with bacterial fermentation broth.

Bacterial and metabolite preparations may be applied in any manner knownfor treating seeds, vegetative propagation units, plants and soil withbacterial strains. Spraying, atomizing, dusting, scattering, pelleting,dipping or pouring may be chosen in accordance with the intendedobjective and the prevailing circumstances. Advantageous rates ofapplication for seed treatment are normally from 10¹¹ to 10¹² cfu/ha andfor spraying 10¹² to 10¹⁴ cfu/ha or a corresponding amount of bacterialmetabolites.

EXAMPLE 1 Isolation of the Microorganism MA 342

The dug up roots of the plant Empetrum nigrum were washed in sterile tapwater to remove adhering soil. From a young root a piece, 2-3 cm long,was cut out and handled under sterile conditions. The piece was takenfrom the region above the root tip area. Small cuts were made in theroot piece with a flamed scalpel. The root piece was then rubbed againstthe surface of TSA 10 agar. After bacteria had grown out, MA 342 waspicked and was pure cultured on to TSA 10.

EXAMPLE 2 Preservation of the Microorganism MA 342

The Pure culture was deep frozen in small ampoules at -70° C. As freezeprotecting agent were used 10% glycerol in tap water, pH adjusted to7.15 after autoclaving. After freezing at -70° C., the ampoules werestored at -20° C.

For long term preservation the isolate was freeze dried. After growingfor 48 hours on TSA 10 agar, the bacterial lawn was scraped off the agarsurface, mixed with a freeze drying protecting agent (50 g Dextran T 70(Pharmacia Fine Chemicals Ltd.); 50 g Na-L-glutamate (Kebo AB) in 1000ml of distilled water), poured into small ampoules (20 ml) and put in aHetosicc freeze drier (Heto Ltd., Denmark) for 24 hours. After freezedrying the ampoules were gas tightly sealed with rubber stoppers andstored at 4° C.

EXAMPLE 3 Effect of MA 342 against Microdochium nivale in primarygreenhouse screenings

The bacterium was applied to the seeds of wheat as follows: 24 hours oldcultures on TSA 10, grown at 15° C., were scraped off from the agarsurface of a 9 cm Petri dish and mixed with 40 ml of nutrient broth(SNB: 18 g sucrose; 5 g bacterial peptone (Oxoid Ltd.); 2 g yeastextract (Oxoid Ltd.); 0.5 g K₂ HPO₄ and 0.25 g MgSO₄.7H₂ O in 1000 mldistilled water and pH adjusted to 7.2-7.4) and 40 ml of a 2% (w/v)solution of sodium carboxymethyl cellulose (CMC) in sterile distilledwater. This mixture was poured over the seeds. After 20 minutes theexcess mixture was poured off and the seeds were dried under a fanovernight.

For each treatment in this greenhouse screening two pots were sown with50 seeds in each pot. The pots were 18 cm in diameter and 4 cm high andfilled to two thirds with an unsterilized commercial peat mixture(Enhetsjord K Normal), mixed with 20% (v/v) sand.

The winter wheat seeds (cv. "Kosack") were artificially infested with M.nivale prior to the treatment with bacteria. The pathogen was cultivatedfor seven days in potato dextrose broth (24 g Potato Dextrose Broth(Difco Ltd.) per 1000 ml distilled water) at room temperature on arotary shaker. The resulting slurry was homogenized with a kitchenblender and poured over the seeds. After 30 minutes the liquid waspoured off and the seeds were left to dry under a fan over night. Seedsthus infested were then treated with MA 342 and sown in pots asdescribed above.

After sowing, the pots were covered with glass lids and placed in thedark at 6° C. After five days the lids were removed, each pot wateredwith 100 ml of water and placed inside a five liters plastic bag thatwas supported by two wooden sticks. The pots were then placed in agreenhouse at 12-15° C. for eight days.

The following treatments of seeds were tested:

1. P. chlororaphis strain MA 342, mixed with SNB/CMC, on M.nivale-infested seeds

2. M. nivale-infested seeds, as disease control

3. Untreated seeds as healthy control

The disease suppressive effect was recorded as the percentages ofemerged, healthy (=without mycelia) plants out of those sown. Resultsfrom a typical M. nivale primary screening is shown in table 1.

                  TABLE 1    ______________________________________    Effect of MA 342 on emergence and disease development in winter    wheat, raised from M. nivale-infested seeds                 Percentage (%)                            Percentage (%) healthy    Treatment    emergence  plants out of those sown    ______________________________________    1. MA 342    87         81    2. Disease control                 13         7    3. Untreated control                 90         89    ______________________________________

EXAMPLE 4 Effect of MA 342 against Drechslera teres in secondarygreenhouse screenings

For these screenings the isolate MA 342 was grown for 48 hours in halfstrength (15 g/l) Tryptic soy broth (Difco Ltd.) on a rotary shaker inthe dark at 18-20° C. Seeds of the barley cultivar "Golf", naturallyinfected with D. teres, were then mixed with 300 ml of the resultingbacterial suspension per kg seed in a plastic bag and, after mixing, thebag was shaken for about 4 minutes. Seeds thus treated were dried undera fan at room temperature for one day and then sown in pots as describedin Example 3.

The sown pots, three per treatment, were placed first in the dark at 6°C. for seven days and then in a greenhouse at about 20° C. as describedin Example 3. For reading treatment effects the frequency of germinatedplants and the frequency of plants with primary attack on the first leafwere counted. The bacterial effect was related to an untreated controland seeds treated with the fungicide Panoctine Plus 400 (guazatine 150g/l+imazalil 10 g/l), Rhone-Poulenc Ltd., in a dosage of 4 ml per kgseed.

                  TABLE 2    ______________________________________    Effect of MA 342 and of Panoctine Plus 400 against D. teres in    barley in a typical greenhouse secondary screening                  Percent germinated                               Percent plants with    Treatment     plants       attack on the first leaf    ______________________________________    Control       92.5         13.0    Panoctine Plus 400, 4 ml/kg                  95.5         0.0    MA 342, 300 ml/kg                  96.1         0.0    ______________________________________

EXAMPLE 5 Effect of MA 342 on plant pathogens in field experiments

Field experiments, designed as randomized blocks and with three to eightrepetitions, had plot sizes varying between experiments from 0.15 m²(one T. caries experiment) to about 15 m² (most experiments). Theexperiments were placed at different localities in Sweden and in mostcases on loamy soils with about 3 percent humus content.

Treatments of seeds with bacteria and with Panoctine Plus 400 were doneas described in Example 4 above. After the treated seeds had been driedwith a fan they were stored at room temperature for various times beforethey were sown in field plots. All seeds, except those infested with T.caries, were naturally infested or infected with the various diseasestested. Seeds of winter wheat (cv. "Kosack") were artificially infestedwith spores of T. caries by mixing 2 g crushed T. caries-ears with 1 kgwheat seeds.

Effect of MA 342 on Tilletia caries

The effect was read as the frequency of infected ears at time ofripening. Results obtained in two trials in 1991/92 and two trials in1992/93 are shown in Table 3. The difference between bacterial treatmentand the fungicide treatment is significant in 1992/93.

                  TABLE 3    ______________________________________    Effect of MA 342 bacterial suspension against seed borne Tilletia    caries infection                    Percent infected ears    Treatment         1991/92 1992/93    ______________________________________    Control           23      65    Panoct. 400*, 4 ml/kg                      2       24    MA 342, 300 mg/kg 0       9    ______________________________________     *Panoctine 400 (guazatine 150 g/l), RhonePoulenc Ltd.

Effect of MA 342 on Drechslera teres, D. raminea, D. avenae and Ustilagoavenae

In the field experiments with these pathogens the number of germinatedplants per m² and the number of infected plants per m² were measuredand, in addition, in most of the experiments also i) grain yield ii)thousand kernel weight and iii) weight per hectolitre.

Effect on Drechslera teres:

Results from field experiments conducted in 1991-1993 and where effectsagainst D. teres-infection in barley were tested are shown in tables 4,5 and 6.

                  TABLE 4    ______________________________________    Results from four field experiments in barley infected with    Drechslera teres in 1991. Means from four experiments conducted at    Alnarp, Lanna, Strangnas and Ultuna             Yield  No. of   Infected                                    Hectolitre                                           1000-kernel    Treatment             kg/ha  plants/m.sup.2                             plants/m.sup.2                                    weight, kg                                           weight, g    ______________________________________    Control  4970   361      47     64.7   41.5    Pan. Plus 400,             5390   353      1      66.3   43.8    4 ml    MA 342, 300             5480   353      1      66.0   43.7    ml/kg    ______________________________________

                  TABLE 5    ______________________________________    Results from five fields experiments in barley infected with Drechs-    lera teres in 1992. Means from experiments conducted at Svalof,    Nygard, Kolback, Ultuna and Robacksdalen             Yield  No. of   Infected                                    Hectolitre                                           1000-kernel    Treatment             kg/ha  plants/m.sup.2                             plants/m.sup.2                                    weight, kg                                           weight, g    ______________________________________    Control  4290   358      48     67.9   51.7    Pan. Plus 400,             4380   378      1      67.8   50.5    4 ml    MA 342, 300             4300   380      1      68.3   52.6    ml/kg    ______________________________________

                  TABLE 6    ______________________________________    Results from two field experiments in barley infected with Drechs-    lera teres in 1993. Means from the experiments conducted at Kol-    back and Ultuna                      Yield  Infected    Treatment         kg/ha  plants/m.sup.2    ______________________________________    Control           6310   74    Pan. Plus 400, 4 ml                      6730   1    MA 342, 200 ml/kg 6720   5    ______________________________________

Effect on Drechslera graminea:

The results from experiments with D. graminea-infected seeds conductedin 1991-1993 are shown in tables 7, 8 and 9.

                  TABLE 7    ______________________________________    Results from one field experiment in 1991 conducted in Uppsala in    barley infected with D. graminea                      Yield  Infected    Treatment         kg/ha  plants/m.sup.2    ______________________________________    Control           3440   31    Pan. Plus 400, 4 ml                      4160   2    MA 342, 300 ml/kg 4390   1    ______________________________________

                  TABLE 8    ______________________________________    Results from five field experiments conducted in 1992 in barley    infected with D. graminea. Means from experiments conducted at    Svalov, Nygard, Kolback, Ultuna and Robacksdalen             Yield  No. of   Infected                                    Hectolitre                                           1000-kernel    Treatment             kg/ha  plants/m.sup.2                             plants/m.sup.2                                    weight, kg                                           weight, g    ______________________________________    Control  2590   383      101    66.2   40.2    Pan. Plus 400,             3470   381      5      66.6   39.9    4 ml    MA 342, 300             3460   368      7      66.2   39.8    ml/kg    ______________________________________

                  TABLE 9    ______________________________________    Results from two field experiments in 1993 in barley infected with    D. graminea. Means from experiments conducted at Kolback and    Ultuna                      Yield  Infected    Treatment         kg/ha  plants/m.sup.2    ______________________________________    Control           2810   46    Pan. Plus 400, 4 ml                      4160   1    MA 342, 200 ml/kg 3990   8    ______________________________________

Effect on Drechslera avenae:

The results from experiments with D. avenae-infected oats seedsconducted in 1991-1993 are shown in tables 10, 11 and 12.

                  TABLE 10    ______________________________________    Results from one field experiment in 1991 conducted in Uppsala in    oats ("Puhti") infected with D. avenae                      Yield  Infected    Treatment         kg/ha  plants/m.sup.2    ______________________________________    Control           4940   74    Pan. Plus 400, 4 ml                      4860   32    MA 342, 300 ml/kg 5090   17    ______________________________________

                  TABLE 11    ______________________________________    Results from four field experiments in 1992 in oats ("Puhti" and    "Vital") infected with D. avenae. Means from experiments conducted    at Svalov and Ultuna             Yield  No. of   Infected                                    Hectolitre                                           1000-kernel    Treatment             kg/ha  plants/m.sup.2                             plants/m.sup.2                                    weight, kg                                           weight, g    ______________________________________    Control  3990   428      22     57.5   35.4    Pan. Plus 400,             4080   456      13     57.8   35.5    4 ml    MA 342, 300             4000   445      3      57.4   35.0    ml/kg    ______________________________________

                  TABLE 12    ______________________________________    Results from a field experiment in 1993 conducted in Uppsala in    oats ("Vital") infected with D. avenae                      Yield  Infected    Treatment         kg/ha  plants/m.sup.2    ______________________________________    Control           7570   79    Pan. Plus 400, 4 ml                      7870   14    MA 342, 200 ml/kg 7680   27    ______________________________________

Effect on Ustilago avenae:

In the field experiments with U. avenae bund ears per m² or percentageof bunt ears were read at the time of ripening. Grain yield was notmeasured. Results from three experiments conducted 1991-1993 are shownin Table 13.

                  TABLE 13    ______________________________________    Results from three field experiments conducted in 1991-1993 in    Uppsala in oats infected with Ustilago avenae                 1991       1992       1993    Treatment    Bunt ears/m.sup.2                            % infected ears                                       Bunt ears/m.sup.2    ______________________________________    Control      7          10,6       95    Panoctine Plus 400, 4 ml                 3          8,7        not tested    MA 342, 300.sup.1) ml/kg                 1          1,7        15    ______________________________________     .sup.1) 300 ml 1991 and 1992; 200 ml in 1993.

EXAMPLE 6 Application of MA 342 to seeds and other plant parts

Applying aqueous mixtures containing MA 342 to seeds.

Bacterial suspensions produced as described in example 3 or as describedin example 4 above were mixed with each of the following substances orcompounds:

Talcum powder (Kebo Lab AB), 48 g/l of bacterial suspension

Bacteriological peptone (Oxoid, Ltd.), 5 g/l of bacterial suspension

Tween 20 (Merck Ltd.), 20 ml/liter of bacterial suspension

Metocel (Cellulose ether, Sveda Kemi AB), 12 g per liter of bacterialsuspension

Lissapol (ICI Agrochemicals Ltd.), 1 g per liter of bacterial suspension

Bond (Newman Agrochemicals Ltd.), 1 g per liter of bacterial suspension

In other experiments the bacterial suspensions were centrifuged at10,000× g for about 10 min. and the resulting pellets were resuspendedin either 0.1 M MgSO₄ or in peptone water (5 g of bacteriologicalpeptone (Oxoid, Ltd.) per liter of tap water).

After thoroughly mixing, the resulting suspensions were applied to seedsas described in example 4 for unmixed bacterial suspensions.

Applying freeze-dried bacteria to plant seeds.

MA 342 bacteria, grown in a shake culture as described in example 4above, were centrifuged and the resulting pellet was resuspended in askim milk solution (200 g skim milk powder, Semper AB, Sweden, per literof sterile distilled water) as a freeze drying protecting agent. Themixture was shell frozen in glass jars and then freeze dried in thesejars for about 48 hours in a Hetosicc freeze drier (Heto Ltd., Denmark).The resulting powder was stored at 4° C. in plastic bags or in plasticflasks with a screw cap until used. For seed application the powder waseither mixed in water or in other aqueous solutions and then applied toseeds as described in example 4 for bacterial suspensions, or it wasmixed with seeds in a dry condition by thoroughly shaking the powder andthe seeds (about 10 g powder per kg seed) in a plastic container.

Pelleting seeds treated with MA 342.

MA 342 bacteria, grown in a shake culture as described in example 4above, were mixed in volumes 1:1 with an adherent (2% w/v aqueoussolution of sodium carboxy-methyl cellulose or 50% w/v aqueous solutionof gummi arabicum). The seeds were treated with this mixture as inexample 4 above. An excess amount of bentonite (Dresser Minerals Inc.)or talcum powder (Kebo Lab AB) was then added to the plastic bag, thebag was inflated and vigorously shaken for a couple of minutes. Afterthis the seeds were spread out on big trays under a fan and allowed todry in room temperature.

Spraying plant shoots with bacterial suspensions.

MA 342 bacteria, grown in a shake culture as described in example 4above, were filled in plastic hand sprayers or in a powered sprayer andthen sprayed on to plant leaves and shoots. In other treatments thebacteria were first centrifuged at about 10.000× g for ten minutes, thepellet was resuspended in tap water and this resulting bacterialsuspension was used for the spraying of plant leaves and shoots.

EXAMPLE 7 Effects of purified metabolites from MA 342 against diseasescaused by Drechslera teres in greenhouse experiments.

The isolate MA 342 was grown for 48 hours in half strength (15 g/l)Tryptic soy broth (Difco Ltd.) on a rotary shaker in the dark at 18-20°C. The resulting bacterial suspension was centrifuged at 48,000 g for 30min. and metabolites in the supernatant was then further purified withSep-pak C 18 cartridges (Waters Associates) as follows:

1. 80 ml of supernatant was added to a Sep-pak activated with 10 mlmethanol.

2. The Sep-pac was washed first with 5 ml 30% ethanol and then with 5 ml40% ethanol.

3. The metabolites were eluted by 5 ml 70% ethanol.

4. The 70% ethanol-eluate was evaporated in a rotation-evaporator untilabout 1.5 ml water solution was left. It was then diluted with tap waterup to a volume of 6.5 ml.

Seeds of the barley cultivar "Golf", naturally infected with D. teres,were submerged in this 6.5 ml water solution of the metabolites for 30minutes and were then sown in pots with 50 seeds per pot. The pots werecovered with glass lids and placed in the dark at 6° C. After nine daysthe lids were removed and the pots were placed in a greenhouse at 15-22°C. for about two weeks. Germinated plants and disease attacks were thenread as described in Example 4 above. As controls were used 1) untreatedseeds and 2) seeds treated with supernatant not purified with Sep-pacand containing MA 342 cells.

                  TABLE 14    ______________________________________    Effect of purified metabolites from MA 342 and supernatant con-    taining MA 342 cells against D. teres in barley in a typical green-    house testing                                  Percent plants                    Percent germinated                                  with attack on    Treatment       plants        the first leaf    ______________________________________    Untreated control                    97,0          21,6    Supernatant with MA 342 cells                    99,0          1,0    Evaporated cell-free eluate                    99,0          0,0    ______________________________________

EXAMPLE 8 Results of comparative tests of MA 342 isolate and 11 otherisolates of Pseudomonas chlororaphis received from different culturecollections

Eleven different non-Swedish isolates of Pseudomonas chlororaphis havingthe designations stated in Table 15 were tested, together with MA 342isolate, for effect against leaf spot disease in barley and for inducingreactions in biochemical tests according to the test system API 20 NE.In addition, the isolates were compared for colony appearance andcrystal formation on agar plates. The 11 non-Swedish isolates were fromdifferent countries and are deposited in four different well-reputedculture collections (Table 15).

Test of disease-inhibiting ability in greenhouse tests

The tests were conducted with Drechslera teres-infected barley, asdescribed in Example 4, and all isolates were tested simultaneously iorder to achieve an adequate comparison. As is evident from Table 15 theresults show that MA 342 is clearly unique in the sense that no one ofthe other isolates tested herein has the property like MA 342 in thiskind of test, viz. the ability to inhibit infection by Drechslera teres.

                  TABLE 15    ______________________________________    Designations, country of origin and effects of MA 342 and 11 other    P. chlororaphis isolates against D. teres-infection in barley in    greenhouse tests                           Effect on leaf spot in greenhouse                           tests. Percentage of infected                           plants after treatment with    Isolate designation               Country of  the isolate in question    ______________________________________    MA 342     Sweden      9    USDA B2075 Czechoslovakia                           53    USDA B1869 New Zealand 60    USDA B14874               USA, Colorado                           56    USDA B14869               USA, Illinois                           58    USDA B1854 USA, Louisiana                           43    NCTC 10686 England     54    NCTC 7357  England     58    DSM 6508   Germany     56    ATCC 9446  USA         61    ATCC 17414 USA         50    ATCC 7811  USA         58    Untreated control      71    ______________________________________

Induction of reactions in biochemical tests according to test system API20 NE

The tests were carried out as described above. The results are shown inTable 16. They show that MA 342 also in this respect is unique can bedifferentiated from the other 11 isolates tested. Some of the isolatestested may not, according to this test, be considered to be centralwithin the species Pseudomonas chlororaphis.

                                      TABLE 16    __________________________________________________________________________    Induced reactions by the different isolates tested in a number of    biochemical tests according to the test system API 20 NE. A "+"    represents a positive reaction and a "-" no/negative reaction.    Property tested              Isolate tested    in API 20 NE              MA B  B  B   B   B  NCTC                                      NCTC                                          DSM                                             ATCC                                                 ATCC                                                     ATCC    test      342                 2075                    1869                       14874                           14869                               1854                                  10686                                      7357                                          6508                                             9446                                                 17414                                                     17811    __________________________________________________________________________    Nitrate reduction              -  -  +  -   -   +  +   +   +  +   +   +    Indole production              -  -  -  -   -   -  -   -   -  -   -   -    Acid from glucose              -  -  -  -   -   -  -   -   -  -   -   -    Arginine dihydrolase              +  +  +  +   -   +  +   +   +  +   +   +    Urease    -  -  -  -   -   -  -   -   -  -   -   -    Esculin hydrolysis              -  -  -  -   -   -  -   -   -  -   -   -    Gelatin hydrolysis              +  +  -  -   -   +  +   +   -  +   +   +    B-galactosidase              -  -  -  -   -   -  -   -   -  -   -   -    Glucose assimilation              +  +  +  +   +   +  +   +   +  +   +   +    Arabinose assimil.              -  +  +  -   -   -  +   -   +  -   -   -    Mannose assimilation              -  +  +  -   +   +  +   +   +  +   +   +    Mannitol assimilation              +  +  +  -   -   +  +   +   +  +   +   +    Glucosamine assimil.              -  +  +  -   -   +  +   +   +  +   +   +    Maltose assimilation              -  -  -  -   -   -  -   -   -  -   -   -    Gluconate assimilation              +  +  +  +   +   +  +   +   +  +   +   +    Caprate assimilation              -  +  +  +   +   +  +   +   +  +   +   +    Adipate assimilation              -  -  -  -   -   +  -   -   -  -   -   -    Malate assimilation              +  +  +  +   +   +  +   +   +  +   +   +    Citrate assimilation              +  +  +  +   +   +  +   +   +  +   +   +    Phenyl-acetate assimil.              -  -  +  +   +   +  +   +   +  -   -   +    Cytochrome oxidase              +  -  +  +   +   +  -   +   +  +   +   +    __________________________________________________________________________

Comparison of colony appearance and crystal formation on agar plates

The isolates were cultured in Petri dishes on TSA 10, as describedabove. We observed small differences in colony appearance between alldifferent isolates but all isolates could not be differentiated in thisway. However, the MA 342 isolate was the only isolate forming typicalhyaline crystals in the agar and could therefore, by this property, bedifferentiated from all other isolates.

What is claimed is:
 1. A biologically pure culture of Pseudomonaschlororaphis strain NCIMB 40616 having the ability to produceantipathogenically active metabolites and mutants of said strain havingall of the identifying characteristics of the strain NCIMB
 40616. 2. Acomposition for controlling plant diseases caused by pathogenic fungi,which comprises as active ingredient the microorganism claimed in claim1 or culture broth of said microorganism which containsantipathogenically active metabolites thereof.
 3. The compositionaccording to claim 2, wherein the pathogen is the fungus Drechslerateres.
 4. The composition according to claim 2, wherein the pathogen isthe fungus Drechslera graminea.
 5. The composition according to claim 2,wherein the pathogen is the fungus Drechslera avenae.
 6. The compositionaccording to claim 2, wherein the pathogen is the fungus Microdochiumnivale.
 7. The composition according to claim 2, wherein the pathogen isthe fungus Tilletia caries.
 8. The composition according to claim 2,wherein the pathogen is the fungus Ustilago avenae.
 9. The compositionaccording to claim 2, wherein the active ingredient is in admixture witha carrier composition acceptable in agricultural practice.
 10. Thecomposition according to claim 9, wherein the active ingredient is inadmixture with a liquid carrier.
 11. The composition according to claim9, wherein the active ingredient is impregnated in a solid porousmaterial.
 12. The composition according to claim 9, which furthercomprises additives that serve as adherents.
 13. The compositionaccording to claim 9, which further comprises a nutrient source.
 14. Amethod of controlling plant diseases caused by pathogenic fungi andcomprising the introduction of an effective dose of an active ingredientinto the environment where the pathogenic fungi is to be inhibited,wherein the active ingredient is the microorganism claimed in claim 1 orculture broth thereof containing antipathogenically active metabolitesor derivates thereof.
 15. The method according to claim 14, wherein theactive ingredient is applied to seeds.
 16. The method according to claim14, wherein the active ingredient is applied to plant vegetativepropagation units.
 17. The method according to claim 14, wherein theactive ingredient is applied to plants.
 18. The method according toclaim 14, wherein the active ingredient is applied to a growing mediumin which a plant is growing or is to be grown.